This application relates generally to mass spectrometry and more particularly to an ultrafast laser system for biological mass spectrometry.
Over the past decade, mass spectrometry (“MS”) has become a key analytical tool for analyzing proteins and metabolites. MS has been used to identify post-translational modifications (“PTMs”) of proteins, which are in some cases the signature of aging processes and malignant disease, making them valuable markers for medical diagnosis. Typically, complex protein mixtures or individual proteins resolved by electrophoretic or chromatographic methods have been traditionally subjected to proteolysis, and then the resultant peptide mixtures were introduced to the mass spectrometer by on-line chromatography. Peptide sequence information was then obtained via subjecting individual ions to fragmentation by collision-induced dissociation (“CID”) tandem mass spectrometry (“MS/MS”). Protein identification was then achieved by database analysis using sophisticated search algorithms (e.g., SEQUEST, Mascot), to correlate the uninterpreted peptide MS/MS spectra with simulated (predicted) product ion spectra derived from peptides of the same mass contained in the available databases. However, the generally limited ability to selectively control or direct the fragmentation reactions of peptide ions during CID-MS/MS towards the formation of structurally informative ‘sequence’ ions (i.e., those resulting from amide peptide bond cleavages) or ‘non-sequence’ ions (i.e., those resulting from cleavage of amino acid side chains that are characteristic of the presence of post translational modifications), placed significant limitations on the application of mass spectrometry and associated methodologies for comprehensive proteome analysis. Recently, several groups have begun to explore the use of laser photo-induced dissociation (“PID”) to access alternative or complementary fragmentation pathways to those observed by conventional collision-induced dissociation. However, these approaches typically did not have bond-selective control over the site of energy absorption from the laser pulse, due to rapid intramolecular vibrational relaxation that occurred prior to bond cleavage, and typically required the presence of a chromophore that was able to absorb energy at the wavelength of the laser to induce fragmentation.
The application of tandem mass spectrometry (“MS/MS”) methods to the identification and characterization of proteolytically derived peptide ions has underpinned the emergent field of proteomics. However, the ability of these conventional approaches to generate sufficient product ions from which the sequence of an unknown peptide can be determined, or to unambiguously characterize the specific site(s) of post-translational modifications within these peptides, was highly dependant on the specific method employed for ion activation, as well as the sequence and charge state of the precursor ion selected for analysis. In practice, collision induced dissociation, whereby energy deposition occurs through ion-molecule collisions followed by internal vibrational energy redistribution prior to dissociation, often resulted in incomplete backbone fragmentation, or the dominant loss of labile groups from side chains containing post-translational modifications such as phosphorylation, particularly for peptides observed at low charge states. Thus, there has been great interest in the development of alternate activation methods, such as surface induced dissociation (“SID”), infrared multiphoton dissociation (“IRMPD”), ultraviolet photodissociation (“UVPD”), electron capture and electron transfer dissociation (“ECD” and “ETD”) and metastable atom dissociation, that yield greater sequence information, and that provide selective control over the fragmentation chemistry independently of the identity of the precursor ion. However, each of these methods suffers from certain limitations. For example, ECD and ETD are applicable only to the analysis of multiply-charged precursor ions, while IRMPD and UVPD efficiencies are dependant on the presence of a suitable chromophore for photon absorption.
In accordance with the present application, one aspect of the system provides a laser and a mass spectrometer. Another aspect of the present application employs a laser emitting a laser beam pulse duration of less than one picosecond into an ion-trap mass spectrometer. A further aspect of the present application provides entrance and exit holes in a mass spectrometer for a laser beam pulse passing therethrough, which advantageously reduces undesired surface charges otherwise possible from misalignment within the mass spectrometer. In yet another aspect of the present application, a femtosecond laser beam pulse causes the ultrafast loss of an election from the charged ions for optional further fragmentation and more detailed mass spectrometry analysis. Another aspect of the present application uses electrospray with mass spectrometry and a shaped laser beam pulse having a duration of less than one picosecond. In still another aspect of the present application, Multiphoton Intrapulse Interference Phase Scan procedures are used to characterize and compensate for undesired characteristics in a laser beam pulse used with an ion-trap mass spectrometer. An additional aspect of the present application includes software instructions which assist in determining whether desired mass spectra information has been obtained, and if not, isolating product ions and then causing another ionization and/or fragmentation process to occur. A method of using a laser system for biological mass spectrometry is also provided. Another method employs emitting a shaped laser pulse at an ionized specimen, further ionizing the ionized specimen by removing at least one electron, isolating the ionized specimen, and then using another supplemental activation step including at least one of fs-LID, CID, SID, IRMPD, UVPD, ECD ETD, Post-Source Decay (“PSD”), Electron Ionization Dissociation (“EID”), Electronic Excitation Dissociation (“EED”), Electron Detachment Dissociation (“EDD”), and/or Metastable Atom-activated Dissociation (“MAD”), in the same equipment.
In order to overcome limitations of conventional devices, the present application provides an advantageous approach to protonated peptide sequence analysis and characterization, involving the use of ultrashort laser pulses for nonergodic energy deposition and multistage dissociation in a quadrupole ion trap mass spectrometer. In one aspect of the present application, peptide solutions in methanol/water/acetic acid are introduced to the mass spectrometer by electrospray ionization, then selected precursor ions are isolated and subjected to MS/MS and MS3 by fs-LID or CID.
The present system significantly improves the structural analysis of modified proteins by the introduction of a femtosecond laser into an ion-trap mass spectrometer. The goal is to take advantage of ultrafast activation, i.e. faster than intramolecular energy redistribution, in order to control the ionization and fragmentation processes. Pulse shaping, in this context, provides in-situ selective fragmentation of specific bonds within a peptide. Binary shaped laser pulses are highly effective in controlling the fragmentation of volatile compounds, and when coupled to an ionization source compatible with the introduction of biomolecules into the gas-phase, provides hitherto unavailable structural information for protein sequencing (proteomics), metabolite recognition (metabolomics), lipid characterization (lipidomics) and target-binding recognition such as protein-ligand, and protein-protein interactions (drug design). A shaped femtosecond laser of the present invention can control the ionization and dissociation processes of isolated ions in the gas-phase due to its ability to deliver energy in a timescale faster than intramolecular energy relaxation. This improves two aspects of biological mass spectrometry: Providing greater sequence coverage than conventional methods such as collision induced dissociation, and improving the analysis of modified proteins by avoiding loss or scrambling of the modification group. The acquisition of reproducible dissociation in the mass spectrometer harnesses the ability to deliver transform limited pulses, i.e., without spectral phase distortions, at the ion-packet within the ion trap of a mass spectrometer.
Simple fragmentation of ions by using short wavelength laser sources in the UV and sometimes in the near UV (400 nm) is well known. For these fragmentation processes to occur it is important for the ion of interest to have at least some portion of its molecular structure include a chromophore or region which by itself has an absorption in the UV-Vis wavelength. In such cases absorption of one or two photons deposits energy in the molecule leads to bond dissociation. The amount of energy will equal that of one or at most two photons which in this case will be less than 10 eV. The drawback to this approach is that short-wavelength laser wavelengths are difficult to generate especially with high energy per pulse. In addition, the molecule or ion must absorb the incident wavelength. It would be advantageous to use an approach that can be used with all molecules and ions without requiring that they absorb the incident wavelength. This approach becomes accessible with ultrafast (preferably less than 1 picosecond and more preferably less than 60 femtosecond) laser pulses of the present disclosure, especially those that have longer wavelengths (from near-infra red 700 nm and longer in the infrared 1 to 2 μm).
The present system's use of ultrafast laser pulses opens a new approach to ion activation. The interaction of an ultrafast laser pulse and an ion is very different from that of a nanosecond laser pulse, especially when the photon energy is much smaller than the ionization potential. In general, ionization of a neutral molecule or further ionization of a trapped ion requires 7-9 eV of energy. This energy can be provided through a nonlinear optical interaction between a long wavelength laser (with energy much smaller than that required for ionization) and the molecule. One may loosely divide the character of the intense-laser nonlinear optical ionization into (a) multi-photon ionization, (b) tunneling ionization and (c) over-the barrier ionization. A Keldysh parameter is used for the classification. A free electron in a laser field makes an oscillating motion at the frequency of the laser. The quiver energy or ponderomotive energy is given by
      U    p    =                              (                                    F              0                                      2              ⁢              ω                                )                2            ∝                        I          0                ⁢                  λ          2                ⁢                                  ⁢        where        ⁢                                  ⁢                  F          0                      =                  (                              I            0                                3.51            ×                          10              16                                      )                    1        /        2            and ω is the angular frequency of the laser electric field, or alternatively I0 and λ are the intensity and the wavelength of the laser field. The Keldysh parameter is proportional to the ratio between the binding energy, EB, of the electron and the ponderomotive energy. It is defined as
      γ    =                                                                  E              B                                                        2            ⁢                          U              P                                          ∝              λ                  -          1                      ,and it is noteworthy that the Keldysh parameter is inversely proportional to the wavelength of the laser.
γ as a function of the intensity and wavelength is then calculated. The multi-photon regime corresponds to the condition where γ>1. In the tunneling regime, scattering with the nuclear center is not important. Instead, the potential barrier formed by the core of the atom or molecule and the electric field of the laser becomes small enough for tunneling to become possible. The electron is pulled off in a field ionization process. Nevertheless, there is a difference between a static field and an oscillating field of the same magnitude. In a static field, a tunneling current will always build up. In an oscillating field, a starting tunneling current is pushed back in the next half cycle, unless it is fast enough to reach the other side of the barrier. It can be shown that the Keldysh parameter is also a measure of the ratio between the laser period and the tunneling time. Thus, when γ≈1 or smaller, the laser field can be treated as quasi-static. Generally, the tunneling formula of Amosov, Delone and Krainov (ADK theory) is considered to be a good approximation to the ionization rate.
For γ<1, ionization is in the over-the-barrier regime. In this case, the electron can escape classically from the potential well. There is, however, no sudden step in the ionization rate at the threshold for over-the-barrier ionization. Instead, the ionization rate continues to grow smoothly and continuously with increasing laser intensity.
Given a certain laser in the laboratory, a minimum value of laser intensity will be required to observe the highly non-linear process involving over-the-barrier ionization; this is the so-called appearance intensity. At somewhat higher laser intensity, the saturation intensity, the ionization rate will have increased so much that the process saturates, i.e. the ionization probability approaches 1. For femtosecond lasers, the over-the-barrier regime is significant. At the classical threshold, the ionization lifetime, i.e. the inverse of the ionization rate, is of the order of 10-100 fs.
Therefore, activation of trapped ions is best achieved by using ultrafast long-wavelength pulses in the present system rather than by using conventional UV-Vis lasers (although certain of the present Claims may not be so limited). The activation proceeds through over-the-barrier ionization. The ion of charge n is ionized to produce a radical ion of charge n+1. The newly created ion can also acquire additional energy which leads to fragmentation. The processes that become available with ultrafast lasers with long wavelengths can be used for (i) altering the charge of trapped ions via removal of electrons and to (ii) fragment trapped ions in a time scale that is much faster than intramolecular vibrational relaxation. Fast fragmentation of ions is desirable when the ions have both strong and weak chemical bonds. Unlike slow fragmentation processes like collision induced dissociation in which there is a thermal or statistical distribution of energy, ultrafast fragmentation prevents the redistribution of energy. In slow fragmentation the weak bonds break preferentially and strong bonds cannot be broken. In contrast, in the fast fragmentation of the present system, strong bonds are broken and weak bonds are left intact. This latter case is important for the analysis of post-translational modifications (“PTM”) of proteins. PTM's have been linked to specific diseases, to aging and as markers for stress. Therefore PTM analysis is beneficial for marker elucidation, for diagnostic purposes, and for monitoring the progression of a disease. It is also noteworthy that over-the-barrier ionization of polyatomic molecules becomes more efficient when circularly polarized femtosecond lasers are used.
Pulse characterization and compression are preferably employed with another aspect of the present invention. With the pulse shaper, the pulse duration is controlled and the pulses are tailored to explore the parameter space that provides the desired level of bond dissociation. Additional advantages and features of the present invention will become apparent from the following description and appended Claims, taken in conjunction with the accompanying drawings.